1. Field of the Invention
The present invention relates to the fields of molecular biology, virology and immunology. More specifically, the present invention provides an attenuated recombinant chimeric chikungunya virus (CHIKV) and discloses its use as vaccines and in serological and diagnostic assays.
2. Description of the Related Art
Chikungunya virus has for decades been an important etiologic agent of human disease in Africa and Asia. However, cases are grossly underestimated because Chikungunya virus infections usually cannot be distinguished clinically from dengue. Recently, Chikungunya virus caused epidemics involving millions of people on islands off the eastern coast of Africa that are popular destinations for European tourists (Schuffenecker et al., 2006), as well as in the Indian subcontinent (Charrel et al., 2007; Kalantri et al., 2006). Further, unlike past epidemics that were usually associated with Aedes aegypti transmission, Ae. albopictus was implicated as the principal mosquito vector in the recent Indian Ocean outbreaks.
Typically, Chikungunya virus causes a severely incapacitating, self-limited disease characterized by fever, rash and severe joint pains: the latter can persist for months. Notably, the recent Indian Ocean outbreaks included many fatal cases, raising the possibility that Chikungunya virus has become more virulent. Chikungunya virus may spread into Western Hemisphere through the movement of infected travelers from Asia and Africa (Charrel et al., 2007) or through introduction of infected mosquitoes carried from epidemic sites in shipping containers. The dramatic spread since 1980 of dengue viruses throughout tropical America, via the same vectors and human hosts, serves as a precedent. If introduced into the New World, Chikungunya virus could cause millions of additional cases of severe and possibly fatal disease.
In addition to its enormous potential as an emerging virus, Chikungunya virus is also underestimated as a potential biological weapon. The 39 documented laboratory infections reported by U.S. Department of Health and Human Services in 1981 strongly suggest that Chikungunya virus is infectious via aerosol route. Chikungunya virus was being weaponized by the United States army when the offensive program was terminated and is considered as a biological weapon by the German and Australian governments.
In general, alphaviruses have plus sense RNA genomes of approximately 11.5 kB that encode 4 nonstructural (nsP1-4) and 3 structural proteins (capsid, E1 and E2 glycoproteins encoded by subgenomic 26S mRNA). The terminal untranslated genome regions (UTR) include repeated sequence elements near the 3′ end that are critical for replication in mosquito cells (Kuhn et al, 1996; Kuhn et al., 1992). Alphaviruses enter cells via receptor-mediated endocytosis (Kielian and Helenius, 1986). The high affinity laminin receptor serves as a mammalian and mosquito cell (in vitro) receptor for Sindbis virus (SINV) (Wang et al., 1992) and VEEV (Ludwig et al., 1996), while other protein receptors for SINV have been identified in mouse neural (Ubol and Griffin, 1991) and chicken cells (Wang et al., 1991). C-type lectins (DC-SIGN, L-SIGN) can function as receptors for infection of dendritic cells (Klimstra et al., 2003).
Following passage in cell culture, alphaviruses adapt to bind to heparan sulfate, which is usually accompanied by attenuation in vivo (Bernard et al., 2000; Byrnes and Griffin, 1998; Byrnes and Griffin, 2000; Klimstra et al., 1998). Genomic RNA is translated by cellular components into a nonstructural polyprotein and is the template for minus-strand RNA synthesis involving nonstructural proteins (nsPs) (Strauss and Straus, 1994). The 26S mRNA is translated as a polyprotein; the capsid protein is cleaved in the cytoplasm and the remaining polyprotein is processed in the secretory pathway to yield the E1 and E2 glycoproteins, which are inserted into the plasma membrane. Following encapsidation of genomic RNA, 70 nm enveloped virions mature when nucleocapsids bud through the plasma membrane (Schlessinger and Schlessinger, 1996; Strauss and Strauss, 1994).
Most alphaviruses replicate in and cause extensive cytopathic effects (CPE) in vertebrate cells in vitro, but infection of mosquito cells usually leads to persistent infection not usually accompanied by cytopathic effects. The mechanisms of alphavirus cytopathogenicity are not fully understood. However, the suppression of host cell gene expression, which allows alphaviruses to compete with cellular metabolism for their replication, is an important component. The alphavirus components responsible for shutdown of cellular gene expression have recently been identified for several members of the genus; Old World viruses SINV (Frolova et al., 2002) and Semliki Forest (Garmashova et al., 2007) use their nsP2 for transcriptional shutoff. However, the New World alphaviruses Eastern (EEEV) (Aguilar et al., 2007) and Venezuelan equine encephalitis (VEEV) (Garmashova et al., 2007) rely on their capsid protein rather than on nsP2. The ability of these capsid proteins to inhibit cellular transcription appears to be controlled by the amino-terminal domain rather than by their protease activity or by their positively charged RNA-binding domains.
Despite their importance as emerging viruses and potential biological weapons, there are no licensed vaccines or therapeutics for alphaviruses. Several experimental human and licensed veterinary vaccines have been produced by inactivation of wild-type or attenuated alphaviruses, but all are poorly immunogenic and require multiple vaccinations and frequent boosters (Tsai et al., 2002). Attenuated strains of Venezuelan equine encephalitis virus (VEEV) (Berge et al., 1961) and Chikungunya virus (Levitt et al., 1986) were developed by cell culture passages of virulent, wild-type strains. The VEEV vaccine, TC-83, has been tested extensively in humans and exhibit high rates of reactogenicity and many people failed to seroconvert (Pittman et al., 1996).
The Chikungunya virus vaccine is attenuated and immunogenic in mice and Rhesus macaques, although these monkeys develop viremia after vaccination (Levitt et al., 1986). The vaccine is also highly immunogenic in humans (Edelman et al., 2000). However, 5 of 59 human vaccines developed transient arthralgia during phase II safety studies. Although the mechanism of reactogenicity for the Venezuelan equine encephalitis virus and Chikungunya virus vaccine strains has not been determined, it is likely that the reversions of attenuating point mutations occur. TC-83, developed by 83 serial cell culture passages has only 2 attenuating mutations (Kinney et al., 1993). The Chikungunya virus vaccine strain, which was developed with only 18 passages probably has similar if not lower number of attenuating mutations. Thus, alphavirus vaccine strains with attenuating point mutations are not stable.
Despite its importance as an emerging arboviral disease and its potential as a biological weapon, the prior art is deficient in safety and immunogenic compositions effective as vaccines that can be used to prevent an individual from infection caused by Chikungunya virus. The present invention fulfills this long-standing need and desire in the art.